Myc deregulation is a hallmark of cancer and promotes tumor aggression across multiple tumor types. When deregulated, levels of the full length Myc (MYC, MYCN, or MYCL) transcription factor are drastically increased, leading to a global remodeling of gene expression that is poorly understood. Here we show evidence for and propose ?Myc enhancer invasion? as a novel mechanism in which excess Myc protein invades distal cis-regulatory enhancers that are not normally bound at physiological Myc levels. As enhancers control tissue specific gene expression, Myc invasion creates aberrant regulatory interactions that drive tumor progression and can be targeted as a therapeutic strategy. Specifically, we have developed and provide evidence for a model of Myc enhancer invasion in which 1) a subset of enhancers contain weak Myc binding motifs that are accessed when Myc is deregulated. 2) Invaded enhancers act as reservoirs for excess Myc binding and drive the Myc responsive transcription of target genes. 3) Tumor specific enhancer landscapes specify the context of Myc enhancer invasion leading to different enhancer invasion responsive genes in different tumors. 4) Enhancer invaded pathways and other transcription factors that form enhancers can be targeted to interdict tumor specific Myc transcriptional control. To investigate this model for Myc enhancer invasion, we propose the following specific aims. 1) To map and model Myc enhancer invasion in neuroblastoma and osteosarcoma in order to identify genomic parameters that predicate enhancer invasion. 2) To investigate transcriptional consequences of Myc enhancer invasion in vitro and in vivo in order to functionally validate that tumor specific Myc enhancer invaded target genes are dynamically and selectively responsive to Myc perturbation. 3) To identify and target oncogenic Myc enhancer regulation to establish proof of concept for the therapeutic targeting of Myc enhancer regulation. We will perform this work in pediatric neuroblastoma and osteosarcoma models and primary tumors. In these diseases, Myc deregulation is associated with high risk disease, metastasis, increased tumor aggression, and poor responsiveness to existing treatments. As no targeted therapies exist for Myc deregulated neuroblastoma or osteosarcoma, there is a critical unmet need for novel strategies to identify dependencies and effectors of Myc deregulation. In our preliminary data, we find that the enhancer transcription factor TWIST1 acts as a co-factor of MYCN enhancer invasion and is a specific dependency of MYCN driven neuroblastoma. These data highlight the utility of the proposed approach to connect mechanistic investigation of Myc transcriptional regulation to novel frameworks for the identification and validation of tumor specific therapeutic targets in Myc driven neuroblastoma and osteosarcoma.